The present invention generally relates to the fields of organ transplantation and reconstructive surgery, and to the new field of Tissue Engineering. It more specifically is a new method and materials for generating tissues requiring a blood vessel supply and other complex components such as a nerve supply, drainage system and lymphatic system.
Organ transplantation, as currently practices, has become a major lifesaving therapy for patients afflicted with disease which destroy vital organs including the heart, liver, lungs, kidney and intestine. However, the shortage of organs needed for transplantation has become critical and continues to worsen. Likewise, every major field of reconstructive surgery reaches the same barrier of tissue shortage. Orthopedic surgery, vascular surgery, cardiac surgery, general surgery, neurosurgery, and the others all share this fundamental problem. Therefore, countless patients suffer as a result.
Over the last twelve years, the new field of tissue engineering has arisen to meet this need. The field brings the expertise of physicians, life scientists and engineers together to solve problems of generating new tissues for transplantation and surgical reconstruction. The initial approaches to this problem were described in the 1980""s. Yannas and Burke (Bell, et al., Science 221,1052 (1981); Burke, et al., Ann Surg 194, 413 (1981)( described methods to generate new tissues in vivo by implanting non-living materials such as modified collagens which are seeded with cells to promote guided regeneration of tissue such as skin. Vacanti and Langer (Langer and Vacanti Science 260, 920 (1993); Vacanti, et al., Materials Research Society 252,367 (1992)) described synthetic fibrous matrices to which tissue specific cells were added in vitro. The matrices are highly porous and allow mass transfer to the cells in vitro and after implantation in vivo. After implantation, new blood vessels grow into the devices to generate a new vascularized tissue. However, the relatively long time course for angiogenesis limits the size of the newly formed tissue.
The field of Tissue Engineering is now maturing and undergoing explosive growth. See, for example, Vacanti and Langer, Lancet 354, 32 (1999); Langer and Vacanti Science 260, 920 (1993); Rennie, J. ed. Special report: The promise of tissue engineering. Scientific American 280, 37 (1999); and Lysaght, et al., Tissue Eng 4, 231 (1998). Virtually every tissue and organ of the body has been studied. Many tissue-engineering technologies are becoming available for human use. See, Lysaght, et al. Tissue Eng 4, 231 (1998); Bell, et al., Science 221,1052 (1981); Burke, et al., Ann Surg 194, 413 (1981); Compton, et al., Laboratory Investigation 60, 600 (1989); Parenteau, et al., Journal of Cellular Biochemistry 45, 24 (1991); Parenteau, et al., Biotechnology and Bioengineering 52, 3 (1996); Purdue, et al., J. Burn Care Rehab 18, 52 (1997); Hansbrough and Franco, Clinical Plastic Surg 25, 407 (1998); Vacanti, et al., Materials Research Society 252,367 (1992).
Over time, several techniques to engineer new living tissue have been studied. Technologies include the use of growth factors to stimulate wound repair and regeneration, techniques of guided tissue regeneration using non-living matrices to guide new tissue development, cell transplantation, and cell transplantation on matrices. More recently, new understanding in stem cell biology has led to studies of populations of primordial cells, stem cells, or embryonic stem cells to use in tissue engineering approaches.
To date, all approaches in tissue engineering have relied on the in-growth of blood vessels into tissue-engineered devices to achieve permanent vascularization. This strategy has worked well for many tissues. However, it falls short for thick, complex tissues such as large vital organs, including liver, kidney, and heart. Techniques using three-dimensional printing technology to achieve ordered arrays of channels have been described to begin to solve this problem. See, for example, Griffith, et al., Ann NY Acad Sci 831, 382 (1997); Langer and Vacanti JP Sci Am 280, 62 (1999).
In parallel to these advances, the rapidly emerging field of MicroElectroMechanical Systems (MEMS) has penetrated a wide array of applications, in areas as diverse as automotives, inertial guidance and navigation, microoptics, chemical and biological sensing, and, most recently, biomedical engineering, Langer and Vacanti Sci Am 280, 62 (1999); McWhorter, et al. xe2x80x9cMicromachining and Trends for the Twenty-First Centuryxe2x80x9d, in Handbook of Microlithography, Micromachining and Microfabrication, ed. P. Rai-Choudhury, (Bellingham, Wash: SPIE Press, 1997). Microfabrication methods for MEMS represent an extension of semiconductor wafer process technology originally developed for the integrated circuit (IC) industry. Control of features down to the submicron level is routinely achieved in IC processing of electrical circuit elements; MEMS technology translates this level of control into mechanical structures at length scales stretching from less than 1 micron to greater than 1 cm. Standard bulk micromachining enables patterns of arbitrary geometry to be imprinted into wafers using a series of subtractive etching methods. Three-dimensional structures can be realized by superposition of these process steps using precise alignment techniques. Several groups (Kourepenis, et al., xe2x80x9cPerformance of MEMS Inertial Sensors,xe2x80x9d Proc. AIAA GNandC Conference, Boston, Mass., 1998; Griffith, et al., Annals of Biomed Eng., 26 (1998); Folch, et al., Biotechnology Progress, 14, 388 (1998)) have used these highly precise silicon arrays to control cell behavior and study gene expression and cell surface interactions. However, this approach is essentially a two-dimensional technology and it has not been apparent that it might be adapted to the generation of thick, three-dimensional tissues.
PCT US96/09344 by Massachusetts Institute of Technology describe a three-dimensional printing process, a form of solid free form fabrication, which builds three-dimensional objects as a series of layers. This process uses polymer powders in layers bound by polymer binders whose geometry is dictated by computer assisted design and manufacture. This technique allows defined internal architectures which could include branching arrays of channels mimicking a vascular supply. However, this technique is limited by the characteristics and chemistry of the particular polymers. Also, it severely limits the types of tissue to be fabricated. Polymer walls do not allow the plasma exchange that is needed at the alveolar capillary wall of the lung.
The object of the present invention is to provide a method and materials for creating complex, living vascularized tissues for organ and tissue replacement, especially complex and/or thick structures, such as liver tissue.
A method and materials to create complex vascularized living tissue in three dimensions from a two-dimension microfabricated mold has been developed. The method involved creating a two dimensional surface having a branching structure etched into the surface. The pattern begins with one or more large channels which serially branch into a large array of channels as small as individual capillaries, then converge to one or more large channels. The etched surface serves a template within a mold formed with the etched surface for the circulation of an individual tissue or organ. Living vascular cells are then seeded onto the mold, where they form living vascular channels based on the pattern etched in the mold. Once formed and sustained by their own matrix, the top of the mold is removed. The organ or tissue specific cells are then added to the etched surface, where they attach and proliferate to form a thin, vascularized sheet of tissue. The tissue can then be gently lifted from the mold using techniques such as fluid flow and other supporting material, as necessary. The tissue can then be systematically folded and compacted into a three-dimensional vascularized structure. This structure can then be implanted into animals or paitents by directly connecting the blood vessels to flow into and out of the device. Immediate perfusion of oxygenated blood occurs, which allows survival and function of the entire living mass.
The design of the branching channels can be constructed by a number of means, such as fractal mathematics which can be converted by computers into two-dimensional arrays of branches and then etched onto wafers. Also, computers can model from live or preserved organ or tissue specimens three dimensional vascular channels, convert to two-dimensional patterns and then help in the reconversion to a three-dimensional living vascularized structure. Techniques for producing the molds include techniques for fabrication of computer chips and microfabrication technologies. Other technologies include laser techniques. The two-dimensional surface of the mold can also be varied to aid in the folding and compacting process. For example, the surface can be changed from planar to folded accordian like. It can be stacked into multiple coverging plates. It could be curvilinear or have multiple projections.
Different types of tissue, or multiple layers of the same type of tissue, can be placed adjacent to each other prior to folding and compacting, to create more complex or larger structures. For example, a tubular system can be layered onto a vascular system to fabricate glomerular tissue and collecting tubules for kidneys. Bile duct tubes can be onlaid over vascularized liver or hepatocyte tissue, to generate a bile duct drainage system. Alveolar or airway tissue can be placed on lung capillaries to make new lung tissue. Nerves or lymphatics can be added using variations of these same general techniques. The two-dimensional surface of the mold can also be varied to aid in the folding and compacting process. For example, the surface can be changed from planar to folded accordian like. It can be stacked into multiple coverging plates. It could be curvilinear or have multiple projections.
Examples of tissues and organs which can be fabricated using these methods include, but are not restricted to, organs currently transplanted such as heart, liver, lung, kidney and intestine. Other tissues such as muscle, bone and breast tissue could also be engineered.